ilvIH operon expression in Escherichia coli requires Lrp binding to two distinct regions of DNA

The leucine-responsive regulatory protein Lrp regulates the expression of a number of operons in Escherichia coli, including the ilvIH operon. Earlier in vitro experiments showed purified Lrp binding to two regions of DNA proximal to the ilvIH promoter, an upstream region (-260 to -190) and a downstream region (-150 to -40). The effect of mutations in these regions on ilvIH promoter expression in vivo led to the proposal that activation of transcription required Lrp binding to downstream sites 3, 4, 5, and 6. Binding of Lrp to upstream sites 1 and 2 seemed to enhance promoter expression but was not absolutely required (Q. Wang and J. M. Calvo, J. Mol. Biol. 229:306-318, 1993). Here we present data that require a reevaluation of the above conclusion. Constructs having either a deletion of DNA or a 100-bp substitution of DNA upstream of position -160 showed no ilvIH promoter activity in vivo. These results unambiguously establish that DNA at or upstream of position -160 is required for ilvIH promoter expression. Together with previous results, we conclude that Lrp bound at downstream sites is necessary but not sufficient for promoter activation. In addition, insertion of 4, 6, 8, or 10 bp between the upstream and downstream regions also resulted in a very strong reduction of in vivo promoter expression, even though the binding of Lrp in vitro was not greatly affected by these mutations. Closer inspection showed that the affinity of Lrp for the upstream region of all of these constructs was about the same but that Lrp bound to the downstream region of the wild-type construct with a higher degree of cooperativity than in the case of the others. These mutations may have reduced promoter activity in vivo by eliminating a binding site for some transcription factor other than Lrp. Alternatively, the small-addition mutations may have affected the geometry of these complexes, preventing either an interaction between Lrps bound at upstream and downstream sites (which might be necessary for promoter expression) or preventing the positioning of Lrp bound at upstream sites for productive interaction with the promoter.